1. Field of the Invention
The present invention relates to an NMR measurement method of investigating a sample having plural domains (such as a solid sample where plural components forming crystallites are mixed, a solid sample consisting of a single kind of molecules but having plural crystal systems intermingled, or a solid sample where crystal components and noncrystalline components are mixed) such that NMR spectra of the individual domains are respectively and separately acquired.
2. Description of Related Art
DOSY (diffusion-ordered spectroscopy) is now described as one example of an NMR measurement method for imaging individual components of a mixture respectively and separately and a method of using the inverse Laplace transform for analysis (see K. F. Morris, C. S. Johnson Jr., Journal of American Chemical Society, Vol. 114, p. 3139-3141 (1992) and K. F. Morris, C. S. Johnson Jr., Journal of American Chemical Society, Vol. 115, pp. 4291-4299 (1992)). DOSY is a method of NMR measurement that has become used in a wide range of fields as NMR instrumentation has been improved in accuracy and software programs for treating NMR data have been improved in recent years. DOSY is an expansion of NMR measurement originally proposed in 1965 by Stejskal and Tanner (E. O. Stejskal, J. E. Tanner, Journal of Chemical Physics, Vol. 42, No. 1, p. 288 (1965)).
If a sample that is a mixture of plural kinds of molecules is investigated by a DOSY experiment, NMR spectra of individual constituent molecules can be acquired respectively and separately by making use of differences in diffusion coefficient among the molecules.
Normally, an NMR spectrum of a mixture is observed as a superimposition of spectral components of individual sample components as shown in FIG. 1. On the other hand, each molecular species has an intrinsic value of molecular diffusion coefficient. Therefore, if NMR peaks in an NMR spectrum of a mixture are classified by molecular diffusion coefficient, then NMR spectra of individual molecular species can be acquired respectively and separately.
Diffusion coefficients are measured by observing NMR spectra plural times while varying the diffusion measurement time. In a normal method of DOSY, as the diffusion measurement time or the diffusion rate is increased, the signal intensity of the NMR spectrum attenuates to a greater extent as shown in FIG. 2. Therefore, the signal intensity of an NMR spectrum attenuates at a higher rate with an increasing diffusion rate in a given diffusion measurement time, and vice versa.
The diffusion coefficient is found by analyzing the decay curve. NMR spectra are separated according to diffusion coefficient using inverse Laplace transform. Sharp peaks appear at the positions of the values of diffusion coefficients by using inverse Laplace transform. This facilitates analyzing the spectrum. FIG. 3 shows an example of the result obtained by performing inverse Laplace transform while taking notice of one peak intensity.
Peaks having signal intensities which are made to show identical attenuation behaviors by diffusion are classified into groups by inverse Laplace transform. The result is shown in FIG. 4, where the spectrum can be separated into a spectral component group of slow diffusion A and a spectral component group of fast diffusion B.
In a DOSY method, spectral components are separated according to diffusion coefficient using inverse Laplace transform. As a result, the spectral components can be separated according to component and observed. This is a method of separation employing the fact that each molecule has an intrinsic value of diffusion coefficient.
Measurement methods of separating plural spectral components by making use of differences in relaxation time are also known besides DOSY. Three of them are next introduced.
(1) A measurement method of separating plural spectral components by making use of differences in longitudinal magnetization relaxation time of 13C (carbon) nuclei.
This is a method of separating NMR signals using differences in longitudinal magnetization relaxation time of 13C nuclei as indices. When a 13C NMR spectrum is observed while varying the relaxation measurement time, signal intensity variations reflect relaxation time variations.
Data obtained by actual measurements on polyethylene is shown in FIG. 5 (quoted from W. L. Earl and D. L. Vander Hart, Macromolecules, Vol. 12, pp. 762-767 (1979)). In the measurements, an inversion recovery method was used. That is, the longitudinal magnetization relaxation time of each 13C carbon nucleus was measured by measuring the time in which an inverted signal recovered. The caption for FIG. 5 reads: “13C spectra at 30° C. displaying the rapid recovery of the noncrystalline component (NCC) resonance centered at 31.7 ppm. The pulse sequence on the carbons is (180°-90°-10 s)I; values of τ are indicated. The protons are continuously irradiated at a low level producing Overhaused (OV) enhancement of the carbon signals to avoid transient Overhauser effects. The weaker crystalline resonance at 34.1 ppm is much attenuated in all of these spectra due to a very long T1C. The T1C of the NCC carbons is 175±25 ms.”
When the relaxation measurement time (τ) was 0.025 s, both of signal of 35 ppm and signal of 31 ppm were inverted. When the relaxation measurement time was then set to 10 s, both signals had positive intensities but their behaviors during this time interval were different.
Although the signal of 31 ppm recovered to a positive intensity in the relaxation measurement time of 0.1 s, the signal of 35 ppm did not recover to a positive intensity until the relaxation measurement time of 1 s. Through this sequence of measurements, the signal of 31 ppm and the signal of 35 ppm can be classified as signals having different relaxation times.
The longitudinal magnetization relaxation times of 13C nuclei are affected more strongly by local modes of motion of molecules. Consequently, it can be said that separation of signals using longitudinal magnetization relaxation times of 13C nuclei is a separation method in which local differences in motion of molecules are reflected.
(2) A measurement method of separating signals by making use of differences in transverse magnetization relaxation time among 1H (hydrogen) nuclei and magnetization transfer from 1H nuclei to 13C nuclei.
In this method, the spectrum is observed while varying the measurement time of the transverse magnetization relaxation time. Prior to the observation, magnetization transfer is done from 1H nuclei to 13C nuclei. The spectrum is observed with the 13C carbon nuclei. In a 1H NMR spectrum, peaks of broad linewidths overlap with each other and are not separated well. On the other hand, in a 13C NMR spectrum, peaks have narrow linewidths and so various peaks can be separated and observed.
Accordingly, magnetization transfer from 1H nuclei to 13C nuclei and subsequent peak observation at 13C nuclei are useful for peak separation. Data about the transverse magnetization relaxation time of 1H nuclei is derived by performing Fourier transform and obtaining a spectrum.
By performing Fourier transform, fast components of the transverse magnetization relaxation appear as peaks of broad linewidths, while slow components appear as peaks of narrow linewidths. The processing consisting of Fourier-transforming time-domain signals and displaying the result as a spectrum follows the conventional procedure of NMR. This processing does not improve signal separation.
FIG. 6A shows a schematic representation of this method of measurement (a) and data obtained by actual measurement (b). Both are taken from the Stejskal and Tanner article. In (a), portion “CP” indicates magnetization transfer. It can be seen from (b) that three peaks are separated and observed in the direction of 13C axis. The caption for FIG. 6A reads: “FIG. 1. Pulse sequence and principle of the heteronuclear 2D WISE-NMR experiment. (a) Basic version with proton evolution, cross polarization (CP), and 13C detection with dipolar decoupling of protons (DD). Typical magnetization decays are sketched. For simplicity, just two components, a ‘rigid’ and a ‘mobile’ one, are considered. At the start of the detection period, the four 180° pulses of the TOSS sequence (not shown here) can be applied to suppress 13C spinning sidebands. (b) Extension by a mixing time before cross polarization. The decrease of the difference between proton magnetization levels by proton spin diffusion during the mixing time is indicated.” The caption for FIG. 6B reads: “FIG. 2. WISE-NMR spectra of PS-b-PDMS (50:50 mol %) for a series of mixing times. (a) Minimum effective tm of 0.5 ms, due to the CP contact time of 1 ms. The PDMS (line near 0 ppm) is highly mobile but does not induce significant mobility in the PS (lines at 40, 127, and 144 ppm; the 1H line width averaged between phenyl and methylene protons is 40 kHz). (b) tm=20 ms. The PDMS within 1 nm from the PS-PDMS interface is detected in the sharp components on the PS signals. (c) Within a mixing time of tm=200 ms, the 1H magnetization is approaching spatial equilibration.”
The peak separation has been accomplished by magnetization transfer from 1H nuclei to 13C nuclei and observation of an NMR spectrum at 13C nuclei. On the other hand, a spectral peak indicated by PDMS in the direction of 1H axis is very narrow, whereas a spectral peak indicated by PS is broad. In this way, peaks can be classified by their linewidth on the 1H axis side.
The transverse magnetization relaxation times of 1H nuclei are affected strongly by local modes of motion of molecules, in the same way as the longitudinal magnetization relaxation times of 13C nuclei. Therefore, it can be said that signal separation relying on transverse magnetization relaxation times of 1H nuclei reflects the differences in local kinetics of molecules.
(3) A method of measuring the longitudinal magnetization relaxation times of 1H nuclei as an NMR spectrum of 13C nuclei by performing magnetization transfer to 13C nuclei.
This method consists of observing the longitudinal magnetization relaxation times of 1H nuclei, then performing magnetization transfer from 1H nuclei to 13C nuclei, and acquiring an NMR spectrum of 13C nuclei as shown in FIG. 7. The results of measurement of the longitudinal magnetization relaxation times of 1H nuclei appear as variations in intensity of a 13C NMR spectrum (M. J. Sullivan and G. E. Maciel, Anal. Chem., Vol. 54, pp. 1615-1623 (1982).
The longitudinal magnetization relaxation times of 1H nuclei are uniform within each individual molecule due to 1H-1H homonuclear spin diffusion. This fact and the measurement methods (1)-(3) above are summarized in detail in K. Schmidt-Rohr and H. W. Spiess in “Multidimensional solid state NMR and polymers,” Academic Press (1994).
When NMR spectra of solution samples consisting of mixtures are acquired, DOSY is most frequently used as mentioned previously because NMR spectra can be separated according to each sample component by the use of DOSY. In DOSY, spectra are separated by employing differences in translational diffusion coefficient among molecules in a solution. However, no translational diffusion occurs in solid samples. Therefore, there is the problem that DOSY cannot be applied to solid samples.
On the other hand, if the method (1) above is used, signals originating from a mixture sample can be separated by making use of differences in longitudinal magnetization relaxation time among 13C nuclei. However, the longitudinal magnetization relaxation times of 13C nuclei reflect local kinetics of molecules and so the separation using this measurement method depends on differences among local kinetics of molecules. Consequently, NMR peaks are not always classified according to molecular species.
In particular, in a case where molecules contain methyl groups, there is the problem that peaks of the methyl groups and other peaks of the same molecule are observed to be separated because the methyl groups show very high mobility.
If the above-described method (2) is used, NMR peaks of a mixture sample can be separated by making use of differences in transverse magnetization relaxation time among 1H nuclei. This method also depends on differences in local kinetics of molecules in the same way as peak separation relying on the longitudinal magnetization relaxation times of 13C nuclei. Consequently, NMR peaks are not always separated according to molecular species.
In particular, in a case where molecules contain methyl groups, there is the problem that peaks of the methyl groups and other peaks of the same molecule are observed to be separated because the methyl groups show very high mobility.
If the above-described method (3) is used, experiments of measurements of the longitudinal magnetization relaxations of 1H nuclei and experiments of magnetization transfer from 1H nuclei to 13C nuclei show that the longitudinal magnetization relaxation times of 1H nuclei can be derived as variations in 13C NMR signal intensity of molecules to which 1H nuclei belong. Normally, this method of measurement is applied only to samples of pure substances. If this method of measurement can be applied to mixture samples, great advantages will be obtained. But this method requires tedious analysis of 13C signal variation.
When spins I (normally, 1H (hydrogen) nuclei) have a uniform longitudinal magnetization relaxation time within each molecule due to spin diffusion, if spectral peaks of the spins I can be classified by longitudinal magnetization relaxation time of the spins I, the spectral peaks of the spins I can be separated according to molecular species.
However, the spectral peaks of the spins I are broadened and made featureless due to spin diffusion. Therefore, it is difficult to separate the spectral peaks of the spins I according to longitudinal magnetization relaxation time of the spins I.
If the peaks can be separated, the obtained spectrum is a featureless spectrum of the spins I having broad peaks. The spectrum has a small amount of information. In order to derive a spectrum having a large amount of information, it is essential to acquire a high-resolution spectrum.